The present invention relates to the isolation of pancreatic islets for transplantation into diabetic patients. In preferred embodiments, the invention presents Advanced Islet Separation Technology, an automated method of islet isolation (separation), an automated process control methodology, process control interface, and an automated apparatus to separate and process pancreatic islets in physiologic process solution utilizing microprocessor control and/or computer control of the process variables and automated apparatus, and the extent of islet separation. The invention may be uniformly applied to islets harvested from animals and mammals, either transgenic or non-transgenic.
The islets of Langerhans, endocrine tissue containing insulin producing beta cells, represent about one to two percent of the total mass of the human pancreas. Separation or isolation of the islets from the connective matrix and remaining exocrine tissue is advantageous and beneficial for laboratory experimentation and transplantation purposes. Islet transplantation is a most promising and minimally physiologically invasive procedure for treatment of type I diabetes mellitus. Transplanting islets rather than complete pancreatic tissue has the distinct advantages of ease of transplantation, and the elimination of the pancreatic exocrine function of the donor tissue involving secretion of digestive enzymes. Liberating islets from pancreatic exocrine tissue is the initial and crucial step that influences islet transplantations. The important objective in islet isolations is to provide sufficient numbers of viable functional and potent islets for transplantation.
Collagenases, metalloendoproteinases that cleave collagen into smaller peptide fragments, are zinc-containing enzymes that require divalent calcium as a cofactor for stabilization and optimal activity. Using traditional collagenases detrimentally affects pancreatic digestion due to the impurities present in the collagenase solutions. Traditional collagenase preparations are concentrated from bacterial (Clostridium histolyticum) culture supernatants. Roche, a manufacturer of molecular biochemicals, states that such collagenase preparations are heterogeneous, containing as many as 30 different enzymes, pigments, cellular debris, and endotoxins. The most significant liabilities of traditional collagenase are variability and endotoxin levels. In traditional collagenase, the primary enzymatic constituent is collagenase, classes I and II. Other proteases found include neutral protease, clostripain, elastase, trypsin, and aminopeptidase.
While larger scale islet separation from human pancreata has become possible with advances in technology, the current techniques cited herein fall short in terms of efficiency, and are inadequate for scale up or mass production in which many donor pancreata are processed at different research and transplantation centers, medical facilities, or commercial locations. In consideration of the lack of donor pancreata, current islet isolation techniques are also inadequate to continuously and repetitively batch process porcine pancreata, or pancreata from animals or mammals, transgenic or non-transgenic, to produce islets for xenotransplantation. In the current techniques cited herein and patents referenced herein exist limitations in the methodologies that may significantly affect the outcome of the islet separation process.
The method of mechanical tissue dissociation with glass marbles, steel balls or other sufficiently dense and solid objects, either by hand or with mechanical shaking (Ricordi shaker), may cause tissue damage and trauma to islets resulting from excessive shear stress during the separation process. While repetitive mechanical agitation and contacting the pancreas with solid objects effects tissue disruption aiding enzymatic digestion, such current practices in standard isolation techniques are subjective, and vary between research facilities and transplantation centers.
Although sonication has been employed to aid pancreatic tissue digestion, one certain limitation in this technique is the ‘static’ water bath that the ‘bagged’ pancreas is placed in. Interestingly, this technique is continued until the pancreas appears ‘cracked’, yet, no mention of the internal temperature of the pancreas is noted. Static digestion by any method offers no means of forced-convective heat transfer to maintain a constant processing temperature (cooling during sonication) of the digesting pancreas or the resulting tissue suspension, by the process solution. It is possible that the internal temperature of a bagged pancreas in such a static system exceeds 37 to 40 degrees C., a temperature considered optimal for functional enzymatic digestion, yet, minimal in thermal shock and deactivation of islets due to elevated temperature. A statically digested pancreas in a bag offers no opportunity to maintain a controlled pancreatic processing temperature. This method presents no opportunity to dilute the tissue suspension, which precludes a real-time method to control, deactivate, or inhibit the digestive enzymes in the processing solution, during islet separation and processing, in the dilution and collection phase.
At the XVIII International Congress of the Transplantation Society, Aug. 27-Sep. 1, 2000 in Rome, Italy, advances in pancreatic islet cell transplantation procedures were reviewed and discussed. Existing limits of transplantation and novel approaches to achieving tolerance were evaluated. It was noted that success of recent transplantations (Edmonton Protocol) might certainly be due to the use of immunosuppression that was not toxic to beta cells. Avoiding the use of corticosteroids, induction therapy with anti-IL-2 antibody, and low-dose tacrolimus and sirolimus maintenance were undoubtedly key factors in non-rejection and continued islet tolerance. The quality of the purified islets also contributed to the success of the transplantations, yet, acquired by tedious and laborious manual methods lacking in process control methodology and neglecting important process variables. Current challenges were also assessed, specifically, the standardization of islet separation technology, and the need to development a standardized, reproducible, and automated method to separate and produce high-quality islet cells.
The background art is characterized by U.S. Pat. Nos. 5,273,904; 5,322,790; 5,377,790; 5,424,209; 5,612,188; 5,834,005; 5,837,738; 5,853,976; 5,879,939; 5,919,703; 5,919,775; and 5,952,215; and U.S. Patent Application No. 2004/0248077; the disclosures of which patents and application are incorporated by reference as if fully set forth herein. The background art is also characterized by the following articles, the disclosures of which are incorporated by reference as if fully set forth herein: Bond and Van Wart, Biochemistry, 23:3077, 1984, and Biochemistry, 23:3085-3091, 1984; D. Scharp, World Journal of Surgery 8:143-151, 1984; Linetsky et al., Transplant Proc., 30(2):345-346, March 1998; Vargas et al., Transplantation, 65(5): 722-727, Mar. 15, 1998; Jahr et al., J. Mol. Medicine (Berlin), 77(1):118-120, January 1999; and Eckhardt et al., J. Mol. Medicine (Berlin), 77(1):123-125, January 1999.
Presently there exists no process control method or device for islet separation that takes into account and crucial process variables that may be controlled to optimize islet isolation while standardizing and automating the islet separation process. Importantly, separation and processing variables have been omitted in background art methods and devices which compromise the reproducibility and repeatability of the islet separation process from location to location. Objectively applying advanced process control methodology disclosed herein and automating the islet isolation process with the process control technology and automated apparatus disclosed herein can optimize the islet separation process.